This work was supported by a research grant MOP 119271 to JMW by the Canadian Institute of Health Research. The authors would like to thank Ms. Yao Fang Tan for her outstanding technical assistance.

NOTE: Toutes les procédures d&#39;animaux étaient conformes aux lignes directrices du ministère de médecine comparée à l&#39;Université de Toronto et de santé bien-être animal. 1. Préparation des tranches hippocampiques <ol><li> Stériliser les instruments suivants en utilisant l&#39;autoclave sec à 125 ° C: Manche de bistouri (# 3) (2), une pince de modèle standard, grande (1), Petit dissecteur ciseaux (angle à l&#39;autre) (1), Micro cuillère (cuillère et plat extrémités de spatule) (1), Micro-spatules (arrondis et arrondis extrémités effilées) (2), pinceau fin (1), polie au feu pipette Pasteur (2), des carrés de gaze, 2 x 2 pouces (5). </li><li> Lorsque stérile, mettre les instruments dans un récipient stérile et de garder couvert jusqu&#39;à utilisation. Immédiatement avant la dissection, immerger les instruments dans une solution d&#39;éthanol à 70%. </li><li> Préparer une plaque de culture à 6 puits avec insert de culture avant de commencer la procédure de dissection par addition de 1 ml de milieu de culture / puits et le stockage de la plaque dans l&#39;incubateur à 35 ° C unee 5% de CO 2. </li></ol> 2. Disposer d&#39;instruments de dissection stérile dans la hotte à flux laminaire <ol><li> Vaporiser la hotte à flux laminaire avec 70% d&#39;éthanol et de supprimer des instruments de dissection stérilisés de l&#39;alcool. Laissez les instruments sécher tout en reposant sur une boîte de Pétri stérile pour éviter tout contact entre l&#39;alcool et le tissu du cerveau disséqué. </li><li> Dépôt 5-7 ml de solution de dissection glacée stérilisé dans deux grandes boîtes de Pétri stériles. Un plat se détendre et de nettoyer la tête (sale), l&#39;autre pour le refroidissement et le rinçage du cerveau creusé (propre). Placer un papier filtre stérilisé dans l&#39;un des couvercles plat de Pétri pour disséquer le cerveau. </li><li> Placez un petit papier filtre stérilisé dans une des petites boîtes de Petri couvercle pour disséquer l&#39;hippocampe. Dépôt 3-5 ml de solution de dissection glacée stérilisé dans deux petites boîtes de Pétri stériles. Un plat tiendra l&#39;hippocampe creusé, une tiendra sections lors de la séparation des sections de l&#39;hippocampesous microscope de dissection. </li><li> Préparer le broyeur de tissus en collant un morceau de papier filtre stérilisé à l&#39;étape de coupe et le montage d&#39;une lame de rasoir stérile. Mouiller le papier filtre avec une solution de dissection stérilisés. </li><li> Vaporiser un sac bio propre avec de l&#39;alcool et le placer dans un écoulement laminaire banc propre. </li></ol> 3. hippocampe Dissection <ol><li> Vaporisez le raton P7 Sprague Dawley éthanol à 70% à l&#39;extérieur du banc de nettoyage à écoulement laminaire et décapiter rapidement l&#39;animal en utilisant de grands ciseaux chirurgicaux stériles à l&#39;intérieur du banc de flux laminaire. Laissez tomber la tête en solution de dissection glacée dans l&#39;une des boîtes de Pétri. </li><li> Dans la boîte de Pétri, rincer le sang et les transférer à la tête de papier filtre stérilisé, de face ventrale rapidement. </li><li> Utilisation du scalpel, couper le long de la surface dorsale dans le plan sagittal afin d&#39;exposer le crâne sous-jacent. Couper à travers la peau, mais pas l&#39;os sous-jacent, qui est doux et facilement pénétrable chez les rats de tson âge. Mettez de côté ce scalpel «sale» et ne pas utiliser sur les tissus du cerveau. </li><li> Utilisation des petits ciseaux de dissecteur (inclinées à l&#39;autre) et des pinces, couper ouvert le crâne long suture sagittale du crâne au bregma, le point anatomique sur le crâne où la suture coronale est coupée perpendiculairement par la suture sagittale. Utilisez une pince pour tirer volets du crâne et loin de la ligne médiane du crâne. </li><li> Placez le micro cuillère sur la face inférieure du cerveau, en dessous du tronc cérébral, de soulever légèrement le cerveau sur le crâne. Soulevez le cerveau pour exposer les nerfs optiques et bulbe olfactif sur la surface de base de cerveau. Coupez ces structures avec de petits ciseaux à détacher entièrement le cerveau du crâne. Retirer et transférer le cerveau intact à l&#39;autre grand plat de Pétri contenant une solution de dissection glacée. </li><li> Utilisation de la micro cuillère, transférer le cerveau à un petit couvercle boîte de Petri contenant du papier filtre stérile. Avec une pipette Pasteur stérile, rincer le cerveau avec quelques gouttes de DISsecting solution pour maintenir le tissu humide. </li><li> En utilisant une lame de scalpel &quot;propre&quot; couper les deux hémisphères à part. Transférer l&#39;hémisphère arrière gauche à la grande boîte de Pétri avec micro cuillère et placez côté pia de l&#39;hémisphère dans une solution de dissection glacée pour une utilisation ultérieure. </li><li> Voir le visage médiale de l&#39;hémisphère droit et identifier le cul de sac de bord, une bande importante de la matière blanche le long du bord médial de l&#39;hippocampe. L&#39;utilisation d&#39;un scalpel stérile, faire une coupe sagittale à travers le cul de sac, mais attention, car seulement 0,5 cm de la pointe de scalpel seront suffisants pour couper le cul de sac. </li><li> Utilisation de deux micro-spatules, supprimer le premier hippocampe de l&#39;hémisphère droit en plaçant la main-spatule droite sur le tronc cérébral et la levée du cortex sus-jacente avec la spatule gauche. Soulevez doucement le cortex pour révéler le ventricule latéral et face médiale de l&#39;hippocampe. Une ligne courbe blanche, la frange, doit maintenant être visible. </li><li> Aligner la courbure de la spatule avec le curvature du fimbria et appuyez doucement sur la spatule sous la frange. Faites glisser la spatule à gauche et rouler le long de l&#39;axe de rostrale-caudale et puis soulevez spatule direction dorsale pour enlever hippocampe. </li><li> Transférer l&#39;hippocampe à un deuxième petit plat 2 Petri avec une solution de dissection glacée. Répétez la même procédure sur l&#39;hémisphère gauche pour retirer l&#39;hippocampe gauche. </li><li> Utilisation d&#39;un micro-spatule, transférer soigneusement la hippocampes à l&#39;étape de découpage de tissu. Disposer les adjacente et parallèle les uns aux autres et perpendiculaires à l&#39;axe de la lame de hachage. Utilisez un pinceau pour positionner le tissu et ajouter quelques gouttes de la solution de dissection sur le dessus de l&#39;hippocampe. </li><li> Coupez le tissu dans 400 um tranches sans se arrêter pour enlever des tranches individuelles (en général, ils ne adhèrent pas à la lame). Après l&#39;ensemble de l&#39;hippocampe a été coupé, utilisez le pinceau pour transférer doucement les sections à un deuxième petit plat 2 Petri avec une solution de dissection. </li><li> Sous annonceissecting microscope, séparer soigneusement les tranches flottantes utilisant amicro-spatule et le pinceau. </li><li> Retirer la plaque de culture pré-préparés avec insert de culture de l&#39;incubateur et le placer dans une hotte à flux laminaire. </li><li> Avec une pipette Pasteur polie au feu, dessiner 4-5 tranches dans la pipette et de transfert des tranches à la surface apicale de la membrane de l&#39;insert de culture. Ensuite, ajuster le positionnement avec un pinceau et laisser de l&#39;espace entre les sections individuelles et la frontière de l&#39;insert de culture. </li><li> Avec une pipette Pasteur stérile, retirez la solution de dissection excès de surface apicale de la membrane. REMARQUE: Lors du retrait de solution éviter de tirer des coupes de tissus dans la pipette. Vous pouvez également utiliser une pipette régulière (P200 ou P1000) avec des embouts de pipette stérilisés pour éliminer lentement solution. </li><li> Placer la plaque de culture par du milieu de culture contenant du sérum et les tranches d&#39;hippocampe de nouveau dans l&#39;incubateur à 35 ° C et 5% de CO 2. NOTE: Si les appels d&#39;expérimentationpour générer des cultures à partir de plusieurs animaux, nettoyer soigneusement le banc de nettoyage à écoulement laminaire entre dissections. Instruments revenir à une solution d&#39;alcool et remplacer tous les boîtes de Pétri, des papiers-filtres et des lames de rasoir stérile avant la deuxième dissection. </li></ol> 4. alimentation et en maintenant organotypiques tranches <ol><li> cultures d&#39;alimentation dans un banc de nettoyage à écoulement laminaire stérile. </li><li> Effectuer la première alimentation des sections de culture 2 jours post-dissection. Aspirer moyenne ancienne de culture en utilisant stérilisée pipette en verre. </li><li> Utilisation stériles de 5 ml pipette sérologique pour ajouter 1 ml de milieu frais, stérile, contenant du sérum dans les puits. </li><li> Replacez délicatement l&#39;insert de culture et de prendre soin d&#39;enlever les bulles d&#39;air qui se sont formées sous la surface de la membrane. </li><li> Après la première tétée, changement de milieu tous les deux jours. </li></ol> 5. en incubation des tranches de tissu avec de la thymidine analogues à l&#39;étiquette du nouveau-né neurones <ol><li> Afin d&#39;étudierla maturation et l&#39;intégration des cellules granulaires du dentate dans l&#39;hippocampe, incuber les tranches organotypiques avec un analogue de la thymidine, comme bromodésoxyuridine (BrdU) ou Chlorodeoxyuridine (CldU). Ici, nous utilisons CldU en raison de sa plus grande solubilité dans une solution saline. </li><li> Après 3DIV, ajouter 1 pl de 10 mg ml solution mère / CldU dans une solution saline à 1 ml de milieu de culture pour une concentration finale de 10 pg / ml. Si BrdU est utilisé, corriger la concentration des différences de poids moléculaire. Ajouter à la moyenne avec CldU puits de culture et incuber le tissu avec un milieu contenant CldU pendant 2 heures à 35 ° C. </li><li> Après 2 heures d&#39;incubation à 35 ° C, éliminer le milieu CldU contenant et le remplacer par un milieu d&#39;alimentation régulière de reprendre horaire d&#39;alimentation normale (décrite ci-dessus). </li></ol> 6. Les tissus Fixation et stockage <ol><li> Établir un calendrier pour l&#39;application de traitements et de fixation des échantillons de tissus avant de commencer les expériences de culture (figure 2B). </li> &lt;li&gt; A un post-dissection de jours prédéterminé, à préparer une hotte de laboratoire, ce qui suit: 10 à 50 ml d&#39;un bêcher contenant 4% de paraformaldehyde (PFA) dans du tampon phosphate salin (PBS); un bécher vide milieu de culture mis au rebut; petite pince; et une pipette de 1 000 ml avec embouts jetables. </li><li> Retirer la plaque (s) de culture provenant de la chambre d&#39;incubation et le transfert d&#39;une hotte de laboratoire. Inclinez les inserts et individuels de la plaque avec une pince. Ensuite, utilisez une pipette pour éliminer le milieu de culture et le transfert dans un bécher d&#39;élimination. Inclinez la plaque de culture à un angle pour aider à assurer que tous les milieu de culture est retiré. </li><li> Terminer la suppression moyenne pour une plaque de culture à la fois. Lorsque moyen a été supprimé, ajouter 1 ml de PFA 4% à chaque puits de culture et de bien sceller la plaque avec du parafilm. Répétez l&#39;opération pour autant de plaques de culture que les plaques nécessaires et de transfert à un réfrigérateur à 4 ° C pendant 24 heures. </li><li> Après 24 heures, préparer le suivant dans une hotte: 10-50 ml bécher contenant 0,1% d&#39;azoture de sodium dans du PBS; un videbécher pour PFA rejetées (suivre les précautions de sécurité lors de l&#39;élimination des substances toxiques); petite pince; et 1000 ml pipette à embouts jetables. </li><li> Suivez la procédure décrite au point 6.4 pour ajouter PFA, mais ajouter 1 ml de PBS avec de l&#39;azoture de sodium à la place. Une fois complètement transféré, les plaques d&#39;étanchéité et pour un usage ultérieur en enveloppant les bords en parafilm et stockage dans un réfrigérateur à 4 ° C. </li></ol> 7. sectionnement tissus pour immunohistochimie <ol><li> Effectuer tissus coupe en utilisant un vibratome. La série d&#39;étapes suivante aider à maximiser le rendement des coupes de tissus utilisables à partir de cultures organotypiques. REMARQUE: Immédiatement après la dissection de l&#39;hippocampe et le placage de tranches, le tissu a une épaisseur d&#39;environ 400 um. Toutefois, après 2 à 3 semaines dans la chambre d&#39;incubation, des tranches de tissu vont commencer à se aplatir, ce qui entraîne une épaisseur de section finale de 150 à 300 um. </li><li> Préparez les éléments suivants à la section tissu cultivé: un bistouri n ° 11 blade et à manipuler, une boîte de Pétri en verre contenant PBS glacé, un micro-dissection pince, et une étape de coupe de vibratome propre au tissu monter. </li><li> Utiliser un scalpel pour couper soigneusement le long du périmètre de la membrane de l&#39;insert circulaire de sorte qu&#39;il peut être détaché du boîtier d&#39;insertion en plastique. Laissez suffisamment d&#39;espace entre la tranche de culture et le scalpel. </li><li> Transférer la membrane insert détaché pour une boîte de Pétri contenant du PBS. Après rinçage dans du PBS, utiliser une pince pour transférer la membrane à l&#39;étape de montage de vibratome. </li><li> Ensuite, utilisez le scalpel pour éliminer l&#39;excès de membrane entourant tranches de culture et de réduire les excès de matériau pour créer des bords nets. Cette étape assure que la membrane est plane et peut facilement adhérer à la surface de coupe. </li><li> Placer 1-2 gouttes de colle sur la scène de coupe de vibratome et diffuser, même couche à l&#39;aide d&#39;une aiguille 22 G. Étendre l&#39;adhésif en une forme rectangulaire avec le long bord parallèle à la lame de coupe de la vibratome. Effectuez cette étaperapidement, pour empêcher l&#39;adhésif de sécher. </li><li> Utilisez une pince pour transférer la membrane garni contenant des tranches d&#39;hippocampe à l&#39;étape de coupe et doucement positionner la membrane sur la colle et de se assurer qu&#39;il n&#39;y a pas de bulles d&#39;air. </li><li> Comme le superglue sèche, transférer la scène vibratome avec la membrane collée revenir à la boîte de Pétri contenant PBS. Préparer la lame de vibratome et contenant un azoture de sodium 48 et de la plaque pour stocker des sections de tissu. </li><li> Utilisez le vibratome pour générer 30 um sections du tissu organotypique de tranche et le transfert à un contenant de l&#39;azoture de sodium 48 de la plaque et pour le stockage et la coloration immunohistochimique suivante. </li><li> Reportez-vous à des protocoles de coloration immunohistochimique 26,29 préexistants. </li></ol>

<ol>
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P., Laskowski, A., Gray, W. P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Examination+of+granule+layer+cell+count,+cell+density,+and+single-pulse+BrdU+incorporation+in+rat+organotypic+hippocampal+slice+cultures+with+respect+to+culture+medium,+septotemporal+position,+and+time+in+vitro.">Examination of granule layer cell count, cell density, and single-pulse BrdU incorporation in rat organotypic hippocampal slice cultures with respect to culture medium, septotemporal position, and time in vitro.</a> The Journal of Comparative Neurology. 497 (3), 397-415 (2006).</li><li>Mielke, J. G., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Cytoskeletal,+synaptic,+and+nuclear+protein+changes+associated+with+rat+interface+organotypic+hippocampal+slice+culture+development.">Cytoskeletal, synaptic, and nuclear protein changes associated with rat interface organotypic hippocampal slice culture development.</a> Developmental Brain Research. 160 (2), 275-286 (2005).</li><li>Fabian-Fine, R., Volknandt, W., Fine, A., Stewart, M. G. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Age-dependent+pre-+and+postsynaptic+distribution+of+AMPA+receptors+at+synapses+in+CA3+stratum+radiatum+of+hippocampal+slice+cultures+compared+with+intact+brain.">Age-dependent pre- and postsynaptic distribution of AMPA receptors at synapses in CA3 stratum radiatum of hippocampal slice cultures compared with intact brain.</a> European Journal of Neuroscience. 12 (10), 3687-3700 (2000).</li><li>Laplagne, D. A., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+neurons+generated+in+the+developing+and+adult+hippocampus.">Functional convergence of neurons generated in the developing and adult hippocampus.</a> PLoS Biology. 4 (12), e409 (2006).</li><li>McDonald, H. Y., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Dynamics+of+neurogenesis+in+the+dentate+gyrus+of+adult+rats.">Dynamics of neurogenesis in the dentate gyrus of adult rats.</a> Neuroscience Letters. 385 (1), 70-75 (2005).</li><li>Stone, S. S., et al. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Functional+convergence+of+developmentally+and+adult-generated+granule+cells+in+dentate+gyrus+circuits+supporting+hippocampus-dependent+memory.">Functional convergence of developmentally and adult-generated granule cells in dentate gyrus circuits supporting hippocampus-dependent memory.</a> Hippocampus. 21 (12), 1348-1362 (2011).</li><li>Wang, S., Scott, B. W., Wojtowicz, J. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Heterogenous+properties+of+dentate+granule+neurons+in+the+adult+rat.">Heterogenous properties of dentate granule neurons in the adult rat.</a> Journal of Neurobiology. 42 (2), 248-257 (2000).</li><li>Wojtowicz, J. M., Kee, N. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=BrdU+assay+for+neurogenesis+in+rodents.">BrdU assay for neurogenesis in rodents.</a> Nature Protocols. 1 (3), 1399-1405 (2006).</li><li>Fritsch, R. S. E. R., Weibel, E. R. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Stereological+Methods,+Vol.+1:+Practical+Methods+for+Biological+Morphometry.">Stereological Methods, Vol. 1: Practical Methods for Biological Morphometry.</a> Zeitschrift f&#252;r Allgemeine Mikrobiologie. 21 (8), 630-630 (1981).</li><li>Bunk, E. C., Konig, H. G., Bonner, H. P., Kirby, B. P., Prehn, J. H. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=NMDA-induced+injury+of+mouse+organotypic+hippocampal+slice+cultures+triggers+delayed+neuroblast+proliferation+in+the+dentate+gyrus:+an+in+vitro+model+for+the+study+of+neural+precursor+cell+proliferation.">NMDA-induced injury of mouse organotypic hippocampal slice cultures triggers delayed neuroblast proliferation in the dentate gyrus: an in vitro model for the study of neural precursor cell proliferation.</a> Brain Research. 1359, 22-32 (2010).</li><li>Kolb, B., Pedersen, B., Ballermann, M., Gibb, R., Whishaw, I. Q. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Embryonic+and+postnatal+injections+of+bromodeoxyuridine+produce+age-dependent+morphological+and+behavioral+abnormalities.">Embryonic and postnatal injections of bromodeoxyuridine produce age-dependent morphological and behavioral abnormalities.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 19 (6), 2337-2346 (1999).</li><li>Morris, S. M. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+genetic+toxicology+of+5-bromodeoxyuridine+in+mammalian+cells.">The genetic toxicology of 5-bromodeoxyuridine in mammalian cells.</a> Mutation Research. 258 (2), 161-188 (1991).</li><li>Bannigan, J., Langman, J. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=The+cellular+effect+of+5-bromodeoxyuridine+on+the+mammalian+embryo.">The cellular effect of 5-bromodeoxyuridine on the mammalian embryo.</a> Journal Of Embryology And Experimental Morphology. 50, 123-135 (1979).</li><li>Breunig, J. J., Arellano, J. I., Macklis, J. D., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Everything+that+glitters+isn't+gold:+a+critical+review+of+postnatal+neural+precursor+analyses.">Everything that glitters isn't gold: a critical review of postnatal neural precursor analyses.</a> Cell Stem Cell. 1 (6), 612-627 (2007).</li><li>Duque, A., Rakic, P. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Different+effects+of+bromodeoxyuridine+and+[3H]thymidine+incorporation+into+DNA+on+cell+proliferation,+position,+and+fate.">Different effects of bromodeoxyuridine and [3H]thymidine incorporation into DNA on cell proliferation, position, and fate.</a> The Journal Of Neuroscience : The Official Journal Of The Society For Neuroscience. 31 (42), 15205-15217 (2011).</li><li>Namba, T., Mochizuki, H., Onodera, M., Namiki, H., Seki, T. <a target="_blank" href="http://www.ncbi.nlm.nih.gov/entrez/query.fcgi?db=PubMed&cmd=Search&doptcmdl=Citation&defaultField=Title+Word&term=Postnatal+neurogenesis+in+hippocampal+slice+cultures:+early+in+vitro+labeling+of+neural+precursor+cells+leads+to+efficient+neuronal+production.">Postnatal neurogenesis in hippocampal slice cultures: early in vitro labeling of neural precursor cells leads to efficient neuronal production.</a> Journal of Neuroscience Research. 85 (8), 1704-1712 (2007).</li></ol>

The authors declare that they have no competing financial interests.

Après CldU (ou BrdU) l&#39;administration, le calendrier d&#39;application des agents pharmacologiques peut être choisi de cibler DGCS nouveau-né pendant particuliers fenêtres développement. Par exemple, un agent hypothétique peut être appliquée pendant la seconde injection semaines post-CldU, qui est proposé pour coïncider avec l&#39;âge de neurones immatures qui sont à un stade de développement où GABA est dépolarisants. Les études futures utilisant ce protocole pourraient adapter l&#39;agent pharmacol...

Adult neurogenesis in the mammalian hippocampus represents a remarkable example of the brain&#8217;s innate capacity for adaptability and plasticity. Dentate granule cells (DGCs) are generated from a renewable pool of neural progenitor cells in the hippocampal dentate gyrus, which is one of the two presently well-characterized neurogenic regions in the mammalian brain, and is thought to be particularly important for learning and memory. The hippocampus is part of the limbic system and has a deep location within the mammalian brain, which makes it a difficult target for precise pharmacological manipulation. Additionally, aberrant neurogenesis has been implicated in conditions, such as epilepsy, schizophrenia, and Alzheimer&#8217;s disease, which has prompted interest in understanding the influence of various pharmacological agents during the maturation and survival of newborn neurons. The distinction between postnatal and adult neurogenesis is blurred and previous studies have shown that many features of in vivo neuronal development in the early postnatal period and adulthood are similar25. Here we emphasize postnatal neurogenesis and suggest possible applications to adult neurogenesis.
Organotypic slice cultures provide an efficient in vitro method for studying various physiological properties of the mammalian hippocampus. The value of slice cultures prepared from rodent brains can be summarized in three main qualities: 1) the protocol is straightforward and requires readily available materials; 2) slice cultures allow for pharmacological studies that eliminate complex variables such as the deep anatomic location of the hippocampus and circumvents the blood brain barrier1; and 3) the well characterized structure of the hippocampus and tri-synaptic circuit is preserved2. Previous investigators have used the organotypic hippocampal culture to study synaptic development and physiology3,4, gliogenesis5-7, ischemic brain damage8,9, neuroprotection and neurorepair10-12 as well as epilepsy13-15.The slice cultures could also provide a useful model system allowing for the monitoring of cell development in conjunction with labeling of cells with green fluorescent protein (GFP) or other vital markers.
Slice cultures have also been previously employed to study postnatal hippocampal neurogenesis16-19, but one important factor in the majority of these studies is the well-characterized degeneration that results from explanting tissue from adult animals after approximately 2 weeks in vitro20,21. For this reason, slice cultures are typically prepared from early post-natal (P5-P10) mice or rat pups, which utilizes the improved viability of early postnatal brain tissue for culturing22. While previous studies have shown that the early postnatal and adult hippocampus differ with regards to synaptic physiology and the expression of specific neuronal subtypes23,24, there is substantial conservation of the choreographed developmental program that newborn dentate granule cells proceed through during maturation25. Additionally, recent studies have suggested that the physiological characteristics of newborn DGCs in culture are very similar to immature neurons in the acute hippocampal slice preparation16.

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			<td height="46" rowspan="2" style="height:47px;width:265px;">Name of Reagent/ Equipment</td>
			<td rowspan="2" style="width:131px;">Company</td>
			<td rowspan="2" style="width:135px;">Catalog Number</td>
			<td rowspan="2" style="width:239px;">Comments/Description</td>
		</tr>
		<tr height="20">
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">5-chloro-2&#39;-deoxyuridine (CldU)</td>
			<td style="width:131px;">MP Biomedicals</td>
			<td align="right" style="width:135px;">105478</td>
			<td style="width:239px;">Hazardous, Carcinogenic</td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Cell culture inserts, 30mm diameter, 0.4&#181;m pore size</td>
			<td style="width:131px;">Thermo scientific&#160;</td>
			<td align="right" style="width:135px;">140660</td>
			<td style="width:239px;">Nuclon delta coating on these inserts provides better tissue adhesion and improves slice quality.</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Conical Centrifuge tubes (sterile)</td>
			<td style="width:131px;">Fisher Scientific</td>
			<td style="width:135px;">14-432-22</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Dissector scissors (angled to side)</td>
			<td style="width:131px;">Fine Science Tools&#160;</td>
			<td style="width:135px;">14082-09</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Minimum essential medium (MEM)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">11095; liquid</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Eclipse Ni-U fluorescent microscope</td>
			<td style="width:131px;">Nikon</td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="80">
			<td height="80" style="height:80px;width:265px;">Glue for tissue</td>
			<td style="width:131px;">Krazy Glue</td>
			<td style="width:135px;">KG585</td>
			<td style="width:239px;">Use minimum amount of glue to achieve adhesion as any tissue exposed to glue will be unusable for IHC.</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Hank&#8217;s Balanced Salt Solution (HBSS) (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">14025-092</td>
			<td style="width:239px;">Store at 4&#176;C</td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Horse Serum Heat Inactivated (500 mL)</td>
			<td style="width:131px;">Gibco</td>
			<td style="width:135px;">16050-122</td>
			<td style="width:239px;">Make 50 mL aliquots and store at -20&#176;C</td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Kimwipes</td>
			<td style="width:131px;">Kimberly-Clarke</td>
			<td style="width:135px;">TW 31KYPBX</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="60">
			<td height="60" style="height:60px;width:265px;">Modified glass pipettes (bottom of Pasteur pipette removed and edge smoothed with Bunsen flame)</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Petri Dish (100mm x 15mm) and (60mm x 15mm)</td>
			<td style="width:131px;">Fisher Brand</td>
			<td style="width:135px;">FB0875712 and FB0875713A</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel blades #11</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10011-00</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Scalpel handle #3</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">10003-12</td>
			<td style="width:239px;"></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Serological Pipettes</td>
			<td style="width:131px;">Sorfa Medical Plastic Co.</td>
			<td style="width:135px;">P8050</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Standard Pattern forceps</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">11000-12</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Sterile vacuum filter</td>
			<td style="width:131px;">Thermo-Scientific</td>
			<td style="width:135px;">565-0020</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Surgical Scissors</td>
			<td style="width:131px;">Fine Science Tools</td>
			<td style="width:135px;">14054-13</td>
			<td></td>
		</tr>
		<tr height="21">
			<td height="21" style="height:21px;width:265px;">Syringe driven filter unit</td>
			<td style="width:131px;">Millipore-Millex</td>
			<td style="width:135px;">SLGP033RS</td>
			<td></td>
		</tr>
		<tr height="40">
			<td height="40" style="height:40px;width:265px;">Tissue chopper with moveable stage</td>
			<td style="width:131px;">Stoelting&#160;</td>
			<td align="right" style="width:135px;">51425</td>
			<td></td>
		</tr>
		<tr height="20">
			<td height="20" style="height:20px;width:265px;">Fine tip paintbrush</td>
			<td style="width:131px;"></td>
			<td style="width:135px;"></td>
			<td></td>
		</tr>
	</tbody>
</table>

organotypic slice cultures for studies of postnatal neurogenesis

Nous décrivons ici une technique pour étudier la neurogenèse postnatale l&#39;hippocampe dans le cerveau de rongeurs en utilisant la technique de culture organotypique. Cette méthode maintient la morphologie caractéristique topographique de l&#39;hippocampe, tout en permettant l&#39;application directe des agents pharmacologiques pour les développement gyrus denté de l&#39;hippocampe. En outre, les cultures tranche peuvent être maintenues jusqu&#39;à quatre semaines et donc, permettent d&#39;étudier le processus de maturation des neurones granulaires nouveau-nés. Trancher les cultures permettent la manipulation pharmacologique efficace des tranches d&#39;hippocampe tout en excluant les variables complexes tels que les incertitudes liées à la localisation anatomique profonde de l&#39;hippocampe ainsi que la barrière hémato-encéphalique. Pour ces raisons, nous avons cherché à optimiser les cultures organotypique spécifiquement pour la recherche de la neurogenèse postnatale.

Déterminer si les cultures organotypiques seraient appropriés pour la recherche de la neurogenèse adulte nécessaires qu&#39;ils remplissent deux critères principaux: 1) que les tranches conserver des caractéristiques morphologiques caractéristiques de tranches d&#39;hippocampe après 10-21 jours in vitro (DIV), et 2) que DGCS nouveau-nés peuvent être quantifiés en utilisant des techniques immunohistochimiques standard habituellement utilisés dans la recherche de la neurogenèse adulte. En ce qui conc...

Watch this Scientific Journal Video about Organotypic Slice Cultures for Studies of Postnatal Neurogenesis at JoVE.com

Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Nous décrivons ici une technique pour étudier la neurogenèse postnatale hippocampe en utilisant la technique de culture organotypique. Cette méthode permet la manipulation in vitro de la neurogenèse adulte et permet l&#39;application directe d&#39;agents pharmacologiques à l&#39;hippocampe en culture.

Trancher les cultures organotypiques d&#39;études de neurogenèse postnatale

Here we describe a technique for studying hippocampal postnatal neurogenesis in the rodent brain using the organotypic slice culture technique. This ...

organotypic-slice-cultures-for-studies-of-postnatal-neurogenesis

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JoVE Journal

Developmental Biology

12.2K vues.  University of Toronto.  Nous décrivons ici une technique pour étudier la neurogenèse postnatale hippocampe en utilisant la technique de culture organotypique. Cette méthode permet la manipulation in vitro de la neurogenèse adulte et permet l'application directe d'agents pharmacologiques à l'hippocampe en culture. 

Vidéo: Organotypic Slice Cultures for Studies of Postnatal Neurogenesis

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical and molecular methods provides information of potential target genes and signaling pathways. To further elucidate specific regulatory mechanisms requires a system allowing the manipulation of only a small number of cells of a specific tissue by either overexpression, ablation or re-introduction of specific genes and follow their fate during development. To achieve this ex utero electroporation of hippocampal structures, especially the dentate gyrus, followed by organotypic slice culture provides such a tool. Using this system to generate mosaic deletions allows determining whether the gene of interest regulates cell-autonomously developmental processes like progenitor cell proliferation or neuronal differentiation. Furthermore it facilitates the rescue of phenotypes by re-introducing the deleted gene or its target genes. In contrast to in utero electroporation the ex utero approach improves the rate of successfully targeting deeper layers of the brain like the dentate gyrus. Overall ex utero electroporation and organotypic slice culture provide a potent tool to study regulatory mechanisms in a semi-native environment mirroring endogenous conditions.

Ex Utero Electroporation and Organotypic Slice Culture of Mouse Hippocampal Tissue

Here we present a protocol providing a tool to examine regulatory mechanisms of specific genes during hippocampal development. Employing ex utero electroporation and organotypic slice culture allows the up- and down-regulation of the expression of genes of interest in single cells and follow their fate during development.

Ex Utero L&#39;électroporation et organotypiques Slice culture de souris hippocampe Tissue

Mouse genetics offers a powerful tool determining the role of specific genes during development. Analyzing the resulting phenotypes by immunohistochemical ...

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Biologie du développement

Generation of Genetically Modified Organotypic Skin Cultures Using Devitalized Human Dermis

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function and physiology of their in vivo tissue counterparts. This is exemplified by organotypic skin cultures which faithfully recapitulates the epidermal differentiation and stratification program. Primary human epidermal keratinocytes are genetically manipulable through retroviruses where genes can be easily overexpressed or knocked down. These genetically modified keratinocytes can then be used to regenerate human epidermis in organotypic skin cultures providing a powerful model to study genetic pathways impacting epidermal growth, differentiation, and disease progression. The protocols presented here describe methods to prepare devitalized human dermis as well as to genetically manipulate primary human keratinocytes in order to generate organotypic skin cultures. Regenerated human skin can be used in downstream applications such as gene expression profiling, immunostaining, and chromatin immunoprecipitations followed by high throughput sequencing. Thus, generation of these genetically modified organotypic skin cultures will allow the determination of genes that are critical for maintaining skin homeostasis.

The goal of this paper is to provide a comprehensive and detailed protocol on how to generate genetically modified human organotypic skin from epidermal keratinocytes and devitalized human dermis.

Génération des cultures organotypiques de peau génétiquement modifiées en utilisant Dévitalisées derme humain

Organotypic cultures allow the reconstitution of a 3D environment critical for cell-cell contact and cell-matrix interactions which mimics the function ...

Generating Primary Fibroblast Cultures from Mouse Ear and Tail Tissues

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell lines. However, primary cell cultures usually have a finite life span and need to be frequently re-established. Fibroblasts are an easily accessible source of primary cells. Here, we discuss a simple and quick experimental procedure to establish primary fibroblast cultures from ears and tails of mice. The protocol can be used to establish primary fibroblast cultures from ears stored at RT for up to 10 days. When the protocol is carefully followed, contaminations are unlikely to occur despite the use of non-sterile tissue stored for extended time in some cases. Fibroblasts proliferate rapidly in culture and can be expanded to substantial numbers before undergoing replicative senescence.

We describe a simple and quick experimental procedure for generating primary fibroblasts from the ears and tails of mice. The procedure does not require special animal training and can be used for the generation of fibroblast cultures from ears stored at RT for up to 10 days.

Cultures Génération fibroblastes primaires de souris oreilles et la queue tissus

Primary cells are derived directly from tissue and are thought to be more representative of the physiological state of cells in vivo than established cell ...

Imaging Cleared Embryonic and Postnatal Hearts at Single-cell Resolution

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, and allow for the study of the cellular and molecular basis of normal and abnormal cardiac morphogenesis. Recent emerging technologies of retrospective single clonal analyses make the study of cardiac morphogenesis at single cell resolution feasible. However, tissue opacity and light scattering of the heart as imaging depth is increased hinder whole-heart imaging at single cell resolution. To overcome these obstacles, a whole-embryo clearing system that can render the heart highly transparent for both illumination and detection must be developed. Fortunately, in the last several years, many methodologies for whole-organism clearing systems such as CLARITY, Scale, SeeDB, ClearT, 3DISCO, CUBIC, DBE, BABB and PACT have been reported. This lab is interested in the cellular and molecular mechanisms of cardiac morphogenesis. Recently, we established single cell lineage tracing via the ROSA26-CreERT2; ROSA26-Confetti system to sparsely label cells during cardiac development. We adapted several whole embryo-clearing methodologies including Scale and CUBIC (clear, unobstructed brain imaging cocktails and computational analysis) to clear the embryo in combination with whole mount staining to image single clones inside the heart. The heart was successfully imaged at single cell resolution. We found that Scale can clear the embryonic heart, but cannot effectively clear the postnatal heart, while CUBIC can clear the postnatal heart, but damages the embryonic heart by dissolving the tissue. The methods described here will permit the study of gene function at a single clone resolution during cardiac morphogenesis, which, in turn, can reveal the cellular and molecular basis of congenital heart defects.

We describe a protocol to volumetrically image fluorescent protein labeled cells deep inside intact embryonic and postnatal hearts. Utilizing tissue-clearing methods in combination with whole mount staining, single fluorescent protein-labeled cells inside an embryonic or postnatal heart can be imaged clearly and accurately.

Imaging Effacé embryonnaire et postnatal coeurs à la résolution unicellulaire

Single clonal tracing and analysis at the whole-heart level can determine cardiac progenitor cell behavior and differentiation during cardiac development, ...

Visualization of Cell Cycle Variations and Determination of Nucleation in Postnatal Cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and cytokinesis) and acytokinetic mitosis (karyokinesis but no cytokinesis). Such atypical cell cycle variations result in polyploid and multinucleated cells rather than in cell division. Therefore, to determine cardiac turnover and regeneration, it is of crucial importance to correctly identify cardiomyocyte nuclei, the number of nuclei per cell, and their cell cycle status. This is especially true for the use of nuclear markers for identifying cell cycle activity, such as thymidine analogues Ki-67, PCNA, or pHH3. Here, we present methods for recognizing cardiomyocytes and their nuclearity and for determining their cell cycle activity. We use two published transgenic systems: the Myh6-H2B-mCh transgenic mouse line, for the unequivocal identification of cardiomyocyte nuclei, and the CAG-eGFP-anillin mouse line, for distinguishing cell division from cell cycle variations. Combined together, these two systems ease the study of cardiac regeneration and plasticity.

To distinguish cell division from cell cycle variations in cardiomyocytes, we present protocols using two transgenic mouse lines: Myh6-H2B-mCh transgenic mice, for the unequivocal identification of cardiomyocyte nuclei, and CAG-eGFP-anillin mice, for distinguishing cell division from cell cycle variations.

Visualisation des variations du cycle cellulaire et détermination de nucléation dans postnatales cardiomyocytes

Cardiomyocytes are prone to variations of the cell cycle, such as endoreduplication (continuing rounds of DNA synthesis without karyokinesis and ...

Dissection and Coronal Slice Preparation of Developing Mouse Pituitary Gland

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate embryonic origins and functions &#8212; the neurohypophysis and the adenohypophysis. The developing mouse pituitary gland is tiny and delicate with an elongated oval shape. A coronal section is preferred to display both the adenohypophysis and neurohypophysis in a single slice of the mouse pituitary.
The goal of this protocol is to achieve proper pituitary coronal sections with well-preserved tissue architectures from developing mice. In this protocol, we describe in detail how to dissect and process pituitary glands properly from developing mice. First, mice are fixed by transcardial perfusion of formaldehyde prior to dissection. Then three different dissecting techniques are applied to obtain intact pituitary glands depending on the age of mice. For fetal mice aged embryonic days (E) 17.5 - 18.5 and neonates up to 4 days, the entire sella regions including the sphenoid bone, gland, and trigeminal nerves are dissected. For pups aged postnatal days (P) 5 - 14, the pituitary glands connected with trigeminal nerves are dissected as a whole. For mice over 3 weeks old, the pituitary glands are carefully dissected free from the surrounding tissues. We also display how to embed the pituitary glands in a proper orientation by using the surrounding tissues as landmarks to obtain satisfying coronal sections. These methods are useful in analyzing histological and developmental features of pituitary glands in developing mice.

We present a protocol to dissect pituitary glands and prepare pituitary coronal sections from developing mice.

Dissection et préparation de tranches coronales de développer la glande pituitaire souris

The pituitary gland or hypophysis is an important endocrine organ secreting hormones essential for homeostasis. It consists of two glands with separate ...

Three-dimensional Organotypic Cultures of Vestibular and Auditory Sensory Organs

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. Moreover, although existing culture techniques allow biochemical perturbations, these methods do not provide a means to study the effects of mechanical force and tissue stiffness during development of the inner ear sensory organs. Here we describe a method for three-dimensional organotypic culture of the intact murine utricle and cochlea that overcomes these limitations. The technique for adjustment of a three-dimensional matrix stiffness described here permits manipulation of the elastic force opposing tissue growth. This method can therefore be used to study the role of mechanical forces during inner ear development. Additionally, the cultures permit virus-mediated gene delivery, which can be used for gain- and loss-of-function experiments. This culture method preserves innate hair cells and supporting cells and serves as a potentially superior alternative to the traditional two-dimensional culture of vestibular and auditory sensory organs.

Three-dimensional organotypic cultures of the murine utricle and cochlea in optically clear collagen I gels preserve innate tissue morphology, allow for mechanical stimulation through adjustment of matrix stiffness, and permit virus-mediated gene delivery.

Culture organotypique en trois dimensions des organes sensoriels vestibulaires et auditives

The sensory organs of the inner ear are challenging to study in mammals due to their inaccessibility to experimental manipulation and optical observation. ...

Single-cell Transcriptomic Analyses of Mouse Pancreatic Endocrine Cells

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, including insulin-secreting &#946; cells, are differentiated from common endocrine progenitors during the embryonic stage. Immature endocrine cells expand via cell proliferation and mature during a long postnatal developmental period. However, the mechanisms underlying these processes are not clearly defined. Single-cell RNA-sequencing is a promising approach for the characterization of distinct cell populations and tracing cell lineage differentiation pathways. Here, we describe a method for the single-cell RNA-sequencing of isolated pancreatic &#946; cells from embryonic, neonatal and postnatal pancreases.

We describe a method for the isolation of endocrine cells from embryonic, neonatal and postnatal pancreases followed by single-cell RNA sequencing. This method allows analyses of pancreatic endocrine lineage development, cell heterogeneity and transcriptomic dynamics.

Analyse transcriptomique unicellulaire de cellules endocrines pancréatiques de souris

Pancreatic endocrine cells, which are clustered in islets, regulate blood glucose stability and energy metabolism. The distinct cell types in islets, ...

Neurogenesis Using P19 Embryonal Carcinoma Cells

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of retinoic acid (RA), the suspension cultured P19 cell line is induced to differentiate into neurons. This phenomenon is extensively investigated as a neurogenesis model in vitro. Therefore, the P19 cell line is very useful for molecular and cellular studies associated with neurogenesis. However, protocols for neuronal differentiation of P19 cell line described in the literature are very complex. The method developed in this study are simple and will play a part in elucidating the molecular mechanisms in neurodevelopmental abnormalities and neurodegenerative diseases.

The P19 mouse embryonic carcinoma cell line (P19 cell line) is widely used for studying the molecular mechanism of neurogenesis with great simplification compared to in vivo analysis. Here, we present a protocol for retinoic acid-induced neurogenesis in the P19 cell line.

Neurogenèse à l'aide de cellules de carcinome embryonnaire P19

The P19 cell line derived from a mouse embryo-derived teratocarcinoma has the ability to differentiate into the three germ layers. In the presence of ...

Real-time Bioluminescence Imaging of Notch Signaling Dynamics during Murine Neurogenesis

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic expression. Notch signaling effector Hes1 and the Notch ligand Delta-like1 (Dll1) are expressed in an oscillatory manner in neural stem/progenitor cells. Because the period of the oscillatory expression of these genes is very short (2 h), it is difficult to monitor their cyclic expression. To examine such rapid changes in the gene expression or protein dynamics, fast response reporters are required. Because of its fast maturation kinetics and high sensitivity, the bioluminescence reporter luciferase is suitable to monitor rapid gene expression changes in living cells. We used a destabilized luciferase reporter for monitoring the promoter activity and a luciferase-fused reporter for visualization of protein dynamics at single cell resolution. These bioluminescence reporters show rapid turnover and generate very weak signals; therefore, we have developed a highly sensitive bioluminescence imaging system to detect such faint signals. These methods enable us to monitor various gene expression dynamics in living cells and tissues, which are important information to help understand the actual cellular states.

Neural stem/progenitor cells exhibit various expression dynamics of Notch signaling components that lead to different outcomes of cellular events. Such dynamic expression can be revealed by real-time monitoring, not by static analysis, using a highly sensitive bioluminescence imaging system that enables visualization of rapid changes in gene expressions.

Imagerie en temps réel de bioluminescence de la dynamique de signalisation d'encoche pendant la neurogenèse de Murine

Notch signaling regulates the maintenance of neural stem/progenitor cells by cell-cell interactions. The components of Notch signaling exhibit dynamic ...